Compounds for protecting hydroxyls and methods for their use

ABSTRACT

A hydrocarbyldithiomethyl-modified compound of the Formula:
 
R 1 —O—CH 2 —S—S—R 2 
 
or a salt thereof wherein R 1  is an organic molecule and R 2  is a hydrocarbyl is useful for protecting and/or blocking hydroxyl groups in organic molecules such as nucleotides. The hydrocarbyldithiomethyl-modified compounds can also be used for chemically synthesizing oligonucleotides and for sequencing nucleic acid compounds.

This application is a continuation of application Ser. No. 09/952,719filed on Sep. 12, 2001, now issued as U.S. Pat. No. 6,639,088, which isa division of application Ser. No. 09/412,171, filed on Oct. 5, 1999,now issued as U.S. Pat. No. 6,309,836.

FIELD OF THE INVENTION

The invention relates to biological chemistry in general. In particular,the invention relates to protecting hydroxyls in organic molecules.

BACKGROUND OF THE INVENTION

Temporary protection or blocking of chemically reactive functions inbiological compounds is an important tool in the field of biologicalchemistry. To this end, researchers have developed a number ofprotecting groups. The vast majority of the known protecting groups,however, are acid or base labile and while there are also protectinggroups that are labile under neutral conditions, most of theseprotecting groups are also somewhat acid and base labile. Greene, T W,“Protective Groups in Organic Synthesis”, publishers Wiley-Interscience(1981). Furthermore, many protecting groups suffer additional synthesis,side-reaction, and/or solubility problems. For example, only a fewprotecting groups applied as a part of a linking system between thesolid phase and the oligonucleotide can withstand all the rigors ofoligonucleotide synthesis and deprotection thereby facilitating thefinal purification of oligonucleotides free of truncated or depurinatedfragments. See “Solid Phase Synthesis,” Kwaitkowski et al., PCTInternational Publication WO 98/08857 (1996). Selective post-syntheticderivatization of oligonucleotides also requires selectively cleavableprotecting groups. See, e.g., Kahl & Greenberg, “Introducing StructuralDiversity in Oligonucleotides via Photolabile, ConvertibleC5-substituted Nucleotides,” J. Am. Chem. Soc., 121(4), 597-604 (1999).

Protecting groups also should be removable. Ideally, the protectinggroup is removable under mild conditions, for example, withoutdisturbing interactions between biomolecules. These types of protectinggroups may be useful for deprotecting oligonucleotides withoutdisturbing interactions between oligo/polynucleotide strands. Forexample, International Publication WO 96/23807 entitled “Novel ChainTerminators, The Use Thereof for Nucleic Acid Sequencing and Synthesisand a Method of their Preparation” discloses methods that usenucleotides that are reversibly blocked at the 3′ hydroxyl group. Thesereversibly blocked nucleotides can be used in sequencing methods where,unlike the well-known Sanger sequencing method that utilizes terminatingdideoxynucleotides, the temporarily 3′-OH-protected intermediates can beconverted into nucleotides having a free 3′-OH that may be furtherextended.

One such sequencing method that uses reversibly blocked nucleotides isknown as Sequencing by Synthesis (SBS). SBS determines the DNA sequenceby incorporating nucleotides and detecting the sequence one base at atime. To effectively sequence long stretches of a nucleic acid usingSBS, it is advantageous to be able to perform multiple iterations of thesingle nucleotide incorporation. Accordingly, SBS-based methods require3′-OH protecting groups that are removable under conditions that do notdistrupt the primer and target DNA interactions. As such, there exists aneed for nucleotide triphosphates that are reversibly blocked at the 3′position and which are also effective substrates for DNA polymerases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a hydrocarbyldithiomethyl-modifiedcompound of the Formula:R¹—O—CH₂—S—S—R²or a salt thereof, wherein R¹ is an organic molecule and R² is ahydrocarbyl. Before undergoing hydrocarbyldithiomethyl-modification, R¹has at least one hydroxyl group, which after modification is in an etherlinkage. In one embodiment, R² further includes a labeling group. Thelabeling group can be any type of labeling group including fluorescentlabeling groups, which can be selected from the group consisting ofbodipy, dansyl, fluorescein, rhodamin, Texas red, Cy 2, Cy 4, and Cy 6.

In another embodiment, R¹—O represents modified or unmodified aminoacids, peptides, proteins, carbohydrates, sterols, ribonucleosides,ribonucleotides, base- and/or sugar-modified ribonucleosides, base-and/or sugar-modified ribonucleotides, deoxyribonucleosides,deoxyribonucleotides, base- and/or sugar-modified deoxyribonucleosides,and base- and/or sugar-modified deoxyribonucleotides. R¹ can have morethan one hydroxyl group and more than one of the hydroxyl groups can bemodified with a hydrocarbyldithiomethyl moiety. For nucleotideembodiments, the hydrocarbyldithiomethyl modification can be at the 2′and/or, 3′, and/or 5′ hydroxyl positions of the R¹—O.

In another embodiment, R² includes a function that modifies the electrondensity of the dithio function, thereby modifying the stability of thedithiol. Such a function may be provided by a chemical group containingelements selected from the group consisting of oxygen, nitrogen, sulfur,and silicon.

In another aspect, the invention provides ahydrocarbyldithiomethyl-modified compound of the Formula:

or a salt thereof, wherein R¹ is H, a protecting group, phosphate,diphosphate, triphosphate, or residue of a nucleic acid, R² is anucleobase, R³ is H, OH, or a protected form of OH; and R⁴, R⁵ and R⁶are together or separately H, hydrocarbyl, or a residue of a solidsupport. Suitable hydrocarbyls for R⁴, R⁵ and R⁶ include methyl, ethyl,isopropylgy, and t-butyl. In one embodiment, R⁴, R⁵ and R⁶ together orseparately further include a labeling group and/or an electron donatingfunction or electron density modifying function. The electron densitymodifying function can be a heteroatom selected from the groupconsisting of oxygen, nitrogen, sulfur, and silicon.

In another aspect, the invention provides a compound of the Formula:

or a salt thereof, wherein R¹ is H, a protecting group, a phosphate,diphosphate, or a triphosphate, or a residue of a nucleic acid, R² isnucleobase, R³ is H or OH, or a protected form of OH, and R⁴ is H orhydrocarbyl. In one embodiment, R⁴ is modified with a labeling group. Inother embodiments, R⁴ includes a derivatizable function, or R⁴ includesnitrogen, or R⁴ is covalently linked to a solid support.

In another aspect, the invention provides a method for modifying anucleoside including the steps of: a) contacting a nucleoside having atleast one hallogenomethyl-modified hydroxyl group with an thiosulfonatecompound thereby forming a thiosulfonated nucleoside; and b) contactingthe thiosulfonated nucleoside with a hydrocarbylthiol compound therebyforming a hydrocarbyldithiomethyl-modified nucleoside. Usefulthiosulfonate compounds include alkylthiosulfonate andarylthiosulfonate.

In one embodiment, the method includes the step of labeling thehydrocarbyldithiomethyl-modified nucleoside.

In another aspect, the invention provides a method for sequencing anucleic acid including the steps of: a) contacting a target nucleic acidwith a primer wherein at least a portion of the primer is complementaryto a portion of the target nucleic acid; b) incorporating ahydrocarbyldithiomethyl-modified nucleotide into the primer; and c)detecting incorporation of the hydrocarbyldithiomethyl-modifiednucleotide, wherein the hydrocarbyldithiomethyl-modified nucleotide iscomplementary to the target nucleic acid at thehydrocarbyldithiomethyl-modified nucleotide's site of incorporation. Inone embodiment, the incorporating step is catalyzed by a DNA polymerase.Useful sequencing methods that may use the method disclosed aboveinclude minisequencing and sequencing by synthesis whether performed inisolation or performed as a sequencing array.

In another aspect, the invention provides a method for sequencing anucleic acid including the steps of: a) contacting a target nucleic acidwith a primer wherein at least a portion of the primer is complementaryto a portion of the target nucleic acid; b) incorporating a firsthydrocarbyldithiomethyl-modified nucleotide into the primer; c)detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide; d) removing thehydrocarbyldithiomethyl group from the first incorporatedhydrocarbyldithiomethyl-modified nucleotide to form a first elongatedprimer having a free hydroxyl group; e) incorporating a secondhydrocarbyldithiomethyl-modified nucleotide into the first elongatedprimer; and f) detecting the second hydrocarbyldithiomethyl-modifiednucleotide, wherein the first hydrocarbyldithiomethyl-modifiednucleotide and the second hydrocarbyldithiomethyl-modified nucleotideare complementary to the target nucleic acid at each nucleotide's siteof incorporation. Following the sequencing method steps once willidentify the sequence of one nucleobase of the target nucleic acid.Repeating the steps can facilitate identifying the sequence of more thanone nucleobase of the target nucleic acid. The conditions of thesequencing method should be such that the primer anneals or hybridizesto the target nucleic acid in a sequence specific manner. In someembodiments the detecting steps are performed before removing thehydrocarbyldithiomethyl group whereas in other embodiments the detectingthe incorporation steps are performed after removing thehydrocarbyldithiomethyl group. In some embodiments, the method isoptimized for implementing the method in a sequencing array.

In another aspect, the invention provides a compound of the Formula:

wherein R¹ is a H, a protecting group, a phosphate, diphosphate, or atriphosphate, or a residue of a nucleic acid; R² is a nucleobase; R⁴, R⁵and R⁶ are together or separately H or hydrocarbyl; and R⁷ is H,H-phosphonate or phosphoramidite.

In another aspect, the invention provides an oligonucleotide synthesissupport of the formula:

wherein R¹ is H, phosphate, diphosphate, triphosphate, or a protectinggroup, R² is a nucleobase, R³ is H, OH, or a protected form of OH, and Zis a group effective for covalent attachment to a solid support, thesolid support being effective for securing an oligonucleotide duringoligonucleotide synthesis. In some embodiments, Z is selected from thegroup consisting of amino, amido, ester, and ether.

In another aspect, the invention provides a method for synthesizing anoligonucleotide including the steps of: a) providing a 5′ protectedfirst nucleoside secured to a solid support through a linker; b)deprotecting the first nucleoside at its 5′ position; c) covalentlybonding another 5′ protected nucleoside to the first nucleotide at the5′ position of the first nucleoside; d) deprotecting the anothernucleoside at its 5′ position; and e) repeating steps c) and d) forincorporating additional protected nucleosides. For this aspect of theinvention, the linker secures the first nucleotide to the solid supportvia a hydrocarbyldithiomethyl bond. This synthesizing method can beoptimized for manufacturing oligonucleotide arrays.

In some embodiments, the oligonucleotide synthesis method is effectivefor inverting the oligonucleotide thereby forming an oligonucleotidehaving a free 3′ hydroxyl and being secured to a solid support viaanother position.

In another aspect, the invention provides a method for synthesizing anoligoribonucleotide including the steps of: a) providing a firstprotected ribonucleoside covalently linked to a solid support; b)covalently linking at least one hydrocarbyldithiomethyl-modifiedribonucleoside to the first ribonucleoside forming anoligoribonucleotide; c) partially de-protecting the oligoribonucleotideunder acidic or basic conditions; and d) contacting theoligoribonucleotide with a reducing agent under neutral conditionsthereby completely de-protecting the oligoribonucleotide, wherein thehydrocarbyldithiomethyl-modified ribonucleoside includes ahydrocarbyldithiomethyl group bound at the 2′ position of thehydrocarbyldithiomethyl-modified ribonucleoside. Such a method iseffective for preventing cleavage or migration of intemucleotidephosphate bonds during deprotection and is also effective for invertingthe oligoribonucleotide thereby forming a solid phase boundoligonucleotide having a free 3′ hydroxyl. In some embodiments, the pHis neutral and can be about 7 or range from about 5 to about 9. In otherembodiments, the first protected ribonucleoside is secured or covalentlylinked to the solid support via a hydrocarbyldithiomethyl bond.

In another aspect, the invention provides a method for sequencing anucleic acid including the steps of: a) providing a primer arrayincluding a plurality of sequencing primers; b) contacting a targetnucleic acid with the primer array thereby forming target-primercomplexes between complementary portions of the sequencing primers andthe target nucleic acid; c) incorporating a firsthydrocarbyldithiomethyl-modified nucleotide into at least one sequencingprimer portion of the target-primer complexes, the firsthydrocarbyldithiomethyl-modified nucleotide being complementary to thetarget nucleic acid; and d) detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide, wherein the firsthydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the first hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. In one embodiment, the methodfurther includes the steps of: e) removing the hydrocarbyldithiomethylgroup from the first incorporated hydrocarbyldithiomethyl-modifiednucleotide to form a first elongated target-primer complex having a free3′ hydroxyl group; f) incorporating a secondhydrocarbyldithiomethyl-modified nucleotide into the first elongatedtarget-primer complex; and g) detecting the secondhydrocarbyldithiomethyl-modified nucleotide, wherein the secondhydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the second hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. As with other methods describedherein, the detecting step can be performed before or after removing ahydrocarbyldithiomethyl moiety. This sequencing method is effective forproducing a plurality of nucleotide sequences wherein the nucleotidesequences correspond to overlapping nucleotide sequences of the targetnucleic acid.

In another aspect, the invention provides a method for sequencing anucleic acid including the steps of: a) providing a target nucleic acidarray including a plurality of target nucleic acids; b) contacting asequencing primer with the target nucleic acids hereby formingtarget-primer complexes between complementary portions of the sequencingprimers and the target nucleic acids; c) incorporating a firstydrocarbyldithiomethyl-modified nucleotide into at least one sequencingprimer portion of the target-primer complexes, the firsthydrocarbyldithiomethyl-modified nucleotide being complementary to thetarget nucleic acid; and d) detecting the incorporation of the firsthydrocarbyldithiomethyl-modified nucleotide, wherein the firsthydrocarbyldithiomethyl-modified nucleotide is complementary to thetarget sequence at the first hydrocarbyldithiomethyl-modifiednucleotide's site of incorporation. As with other methods describedherein, the detecting step can be performed before or after removing ahydrocarbyldithiomethyl moiety. This sequencing method is effective forproducing a plurality of nucleotide sequences wherein the nucleotidesequences correspond to overlapping nucleotide sequences of the targetnucleic acid.

In another aspect, the invention provides a method for synthesizing anoligonucleotide that includes the steps of: a) providing a 5′ protectedfirst nucleoside covalently bonded to a solid support through ahydrocarbyldithiomethyl containing linker; b) deprotecting the firstnucleoside at its 5′ position; c) covalently bonding another 5′protected nucleoside to the first nucleotide at the 5′ position of thefirst nucleoside; d) deprotecting the another nucleoside at its 5′position; e) optionally repeating steps c) and d) for adding additionalprotected nucleosides thereby producing an oligonucleotide; f)optionally selectively cleaving a protecting group from theoligonucleotide thereby forming a partially deprotected oligonucleotide;g) selectively cleaving the hydrocarbyldithiomethyl containing linker;and h) isolating the partially deprotected oligonucleotide. In oneembodiment, the method further includes the step of modifying the 3′terminus with a reactive or detectable moiety. In another embodiment, atleast one of the 5′ protected nucleosides contains ahydrocarbyldithiomethyl moiety.

Advantages of the invention include introducing temporary or reversiblemutations in proteins, facilitating continuous sequencing methods,blocking reactive species during chemical syntheses, masking chemicalgroups for manufacturing purposes, using hydroxyl groups to introducelabeling groups into organic molecules, and other similar uses. It is tobe understood that particular embodiments of the invention describedherein may be interchanged with other embodiments of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The present invention relates to hydrocarbyldithiomethyl-modifiedcompounds having modified and/or protected hydroxyl groups, methods formanufacturing such compounds, and methods for their use. The generalformula for the hydroxyl-modifying moiety contains a dithiol. Thedithiol is designed so that modified hydroxyls can be de-protected underneutral conditions using mild reducing agents.

A hydrocarbyldithiomethyl-modified compound is shown in Formula 1:R¹—O—CH₂—S—S—R²  (Formula 1)wherein R¹ represents any organic molecule that had at least one freehydroxyl group before undergoing the hydrocarbyldithiomethylmodification. In Formula 1, “O” is the oxygen atom of the hydroxylgroup, which is now protected in an ether linkage. For example, beforemodification, R¹ can be a modified or unmodified amino acid or analogthereof, oligonucleotide, peptide, protein, carbohydrate,deoxyribonucleoside, deoxyribonucleotide, ribonucleoside,ribonucleotide, base- and/or sugar-modified ribonucleoside, base- and/orsugar-modified deoxyribonucleoside, base- and/or sugar-modifiednucleotide, sterol or steroid, as long as the organic molecule selectedhas at least one hydroxyl group capable of being hydrocarbyldithiomethylmodified. As referred to herein, oligonucleotides refers to anynucleotide polymer including polymers of deoxyribonucleotides,ribonucleotides, nucleotide analogs and mixtures thereof.

When R¹—O is an organic molecule having more than one free hydroxylgroup, any number of the free hydroxyls may be modified with ahydrocarbyldithiomethyl moiety. Alternatively, one or more of theadditional hydroxyl groups can be modified and/or protected with otherknown hydroxyl modifying compounds or left unmodified. Differentprotecting groups may be used to protect different hydroxyl groups.Useful protecting groups, other than the hydrocarbyldithiomethyl-basedgroups described herein, and methods for their use are known to those ofskill in the art and include fluorenylmethyloxycarbonyl (FMOC),4-(anisyl)diphenylmethyltrityl (MMTr), dimethoxytrityl (DMTr),monomethoxytrityl, trityl (Tr), benzoyl (Bz), isobutyryl (ib), pixyl(pi), ter-butyl-dimethylsilyl (TBMS), and1-(2-fluorophenyl)-4-methoxypiperidin 4-yl (FPMP). See, e.g., Greene, TW, “Protective Groups in Organic Synthesis”, publishersWiley-Interscience (1981); Beaucage & Iyer, “Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach,” Tetrahedron,48(12):2223-2311 (1992); Beaucage & Iyer, “The Synthesis of SpecificRibonucleotides and Unrelated Phosphorylated Biomolecules by thePhosphoramidite Method,” Tetrahedron, 49(46):10441-10488 (1993); andScaringe et al., “Novel RNA Synthesis Method Using5′-O-silyl-2′-O-orthoester Protecting Groups,” J. Am. Chem. Soc.,120:11820-21 (1998). The choice of protective group can be dictated bythe type of organic molecule to be protected and the methods employed.Therefore, different organic molecules such as peptides,oligonucleotides, carbohydrates, and steroids may each use differentprotective groups. A hydroxyl with a known protecting group or ahydrocarbyldithiomethyl moiety attached to it can be referred to as aprotected form of the hydroxyl.

In Formula 1, R² represents a hydrocarbyl group. As used herein,hydrocarbyl groups include any organic radical having a carbon atomdirectly attached to the remainder of the molecule, e.g., saturated andunsaturated hydrocarbons, straight- and branched-chain aliphatichydrocarbons, cyclic hydrocarbons, aromatic hydrocarbons, heterocyclichydrocarbons, heteroaromatic hydrocarbons, and substituted hydrocarbonssuch as hydrocarbons containing heteroatoms and/or other functionalmodifying groups. The hydrocarbyl group may be covalently linked to asolid support (described below), labeling group or another organicmolecule.

Suitable hydrocarbons include alkyls (e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl, and heptadecyl); alkoxy; alkenyl; C₃₋₈ alkenyloxy; alkynyl;alkynyloxy; C₃₋₂₀ cycloalkyl (e.g. cyclopropyl, cyclobutyl orcyclopentyl) in which the cycloalkyl may be substitutedn by one or morehydrocarbyls or heteroatoms; C₃₋₈ cycloalkoxy (e.g. cyclopentoxy); C₄₋₈cycloalkenyloxy (e.g. cyclopenten-3-yloxy); aryl (e.g. phenyl) oraralkyl (e.g. benzyl) in which the aryl may be substituted with one ormore C₁₋₄ alkyl, halogen, hydroxy, C₁₋₄ alkoxy, amino or nitro; aryloxy(e.g. phenoxy); arylalkoxy (e.g. benzyloxy) in which the aryl may besubstituted with one or more C₁₋₄ alkyl, halogen, hydroxy, C₁₋₄ alkoxy,amino or nitro; C₁₋₆ hydroxyalkyl (e.g. hydroxyethyl); and C₁₋₆alkoxyalkyl (e.g. methoxyethyl). In addition, all iso, sec and tertisomers of the aliphatic hydrocarbons are included such as isopropyl andt-butyl.

Substituted hydrocarbon groups are hydrocarbons containingnon-hydrocarbon substituents. Suitable substituents include oxygen,nitrogen, sulfur, phosphorous, halogens (e.g., bromine, chlorine,iodine, and fluorine), hydroxy, carbalkoxy (especially lower carbalkoxy)and alkoxy (especially lower alkoxy), the term, “lower” denoting groupscontaining 7 or less carbon atoms.

Other functional modifying groups capable of moderating the reactivityor lability of the disulfide bond or facilitate synthesizing compoundsof Formula 1 can be incorporated into R². Useful functional modifyinggroups are known and include heteroatoms such as oxygen, nitrogen,sulfur, phosphorous, and halogens. Functional modifying groups alsoinclude heterogroups such as amino, nitro, and cyano. These groups mayfunction as an electron withdrawing or donating groups. Skilled artisansknow whether electron withdrawing or donating groups would beappropriate.

R² may further include a labeling group. Useful labeling groups areknown to those of ordinary skill in the art and include radioactivelylabeled groups, luminescent groups, electroluminescent groups,fluorescent groups, and groups that absorb visible or infrared light.Examples of useful fluorescent labels include bodipy, dansyl,fluorescein, rhodamin, Texas red, Cy 2, Cy 4, and Cy 6. Additionaluseful labels can be found in the “Handbook of Fluorescent probes andResearch Chemicals,” by Richard P. Haugland and “Nonisotopic DNA ProbeTechniques,” Ed. Larry J. Kricka (Academic Press, Inc. 1992).Hydrocarbyldithiomethyl-modified compounds can be created from availablecompounds using the following illustrative method. A iree hydroxyl on ahydroxyl-containing molecule is modified to form a methylthiomethylether. The methylthiomethyl ether can be formed by reacting the hydroxylwith a mixture of acetic anhydride, acetic acid and dimethyl sulfoxide(DMSO). See Hovinen et al., “Synthesis of3′-O-(ω-Aminoalkoxymethyl)thymidine 5′-Triphosphates, Terminators of DNAsynthesis that Enable 3′-Labeling,” J. Chem. Soc. Perkin Trans. I, pp.211-217 (1994). It is to be understood that, if the molecule to bemodified contains more than one hydroxyl group, it may be necessary tofirst protect or block one or more hydroxyl groups that are not to behydrocarbyldithiomethyl-modified.

The methylthiomethyl ether-derivatized compound is then converted to amore reactive species such as a halogenatedmethyl ether. Useful halogensinclude bromine, chlorine, and iodine. The halogenation step can becarried out using any method including treating the methylthiomethylether with N-bromosuccinimide (NBS), or Br₂ in dry chlorethane, orSOCl₂, or N-iodosuccinimide (NIS).

The halogenated methyl ether compound is then converted to ahydrocarbylthiolsulfonate reagent by treating it with an alkylhydrocarbylthiolsulfonate. See Bruice & Kenyon, “Novel AlkylAlkanethiolsulfonate Sulfhydryl reagents, Modification of Derivatives ofL-Cysteine,” J. Protein Chem., 1(1):47-58 (1982) and Plettner et al., “ACombinatorial Approach to Chemical Modification of Subtilisin Bacilluslentus,” Bioorganic & Medicinal Chem. Lett. 8, pp. 2291-96 (1998).

Contacting the hydrocarbylthiolsulfonate reagent with any unsubstitutedor substituted thiol can cause displacement of the sulfonyl moietythereby creating a hydrocarbyldithiomethyl-modified compound. Usefulthiols include branched- and straight-chain aliphatic thiols, aromaticthiols, heteroaromatic thiols, substituted aliphatic thiols,functionally modified thiols, and fluorophore labeled thiols.Functionally modified thiols include thiols substituted at a carbon atomwith an atom or group capable of altering the reactivity of a dithiomoiety, capable of facilitating subsequent labeling, or capable offacilitating immobilization of the modified compound. Useful examples ofmodifying groups include amino, amido, hydroxyl, silyl, cyano,carboxylic esters, or other carboxylic substitutions.

It may be particularly useful to use the compounds of Formula 1 when R¹contains a nucleobase. As used herein, nucleobase includes any naturalnucleobase, synthetic nucleobase, and/or analog thereof. Naturalnucleobases include adenine, guanine, cytosine, thymine, uracil,xanthine, hypoxanthine, and 2-aminopurine. Synthetic nucleobases aretypically chemically synthesized and are analogues of the naturalnucleobases. Synthetic nucleobases are capable of interacting orhydrogen bonding with other nucleobases. Nucleobase containing compoundscan include both nucleosides and nucleotides. Nucleosides andnucleotides can be modified at the 5′, 3′ and/or 2′ hydroxyl positions.Known methods for protecting the 5′, 3′ and/or 2′ positions may be usedin conjunction with the methods described herein to modify individualhydroxyl positions.

For example, the compound shown in Formula 2

or a salt thereof can be synthesized using the methods described herein.In Formula 2, R¹ is H, a protecting group, phosphate, diphosphate,triphosphate, or a residue of a nucleic acid; R² is a nucleobase; R³ isH, OH, or a protected form of OH; R⁴, R⁵ and R⁶ are together orseparately H, hydrocarbyl, or a residue of a solid support. For example,R⁴, R⁵ and R⁶ include together or separately H, methyl, ethyl,isopropyl, t-butyl, phenyl, or benzyl. It may be useful to include asubstituted hydrocarbon having an electron density modifying groupcontaining a heteroatom or other functional modifying group at positionsR⁴, R⁵ or R⁶. For example, R⁴, R⁵ or R⁶ could be methyleneamine,ethyleneamine, or contain an amino group. As an optional aspect, R⁴, R⁵or R⁶ can be modified with a labeling group.

Another illustrative example of useful hydrocarbyldithiomethyl-modifiedcompounds include the compounds of Formula 3

or a salt thereof, wherein R¹ is H, a protecting group, a phosphategroup, diphosphate group, or a triphosphate group; R² is nucleobase; R³is H or OH, or a protected form of OH; and R⁴ is H , a heteroatom, aheterogroup, hydrocarbyl or a label-modified hydrocarbyl. R⁴ may be usedto link the compound of Formula 3 to a solid support.

A nucleobase containing hydrocarbyldithiomethyl-modified compound can bechemically synthesized using the methods described herein. For example:

Compound A (wherein R¹ is a suitable protecting group, R² is anucleobase, and R³ is either a protected hydroxyl or H) is treated witha mixture of DMSO, acetic acid, and acetic anhydride to form amethylthiomethyl ether (compound B)

The methylthiomethyl ether (compound B) is converted to a more reactivehalogenated species (compound C) wherein X is Br, Cl, or I.

Compound C is treated with an alkyl- or arylthiosulfonate such asmethylphenylthiosulfonate (MePhSO₂SH) to prepare compound D.

Compound D is treated with a hydrocarbylthiol compound such as2-thio-aminoethane to form a hydrocarbyldithiomethyl-modified nucleobase(compound E). In some instances, compound E may be the final product.

When the thiol used to form compound E contains a modifiablesubstituent, compound E can be further modified or labeled as shownbelow. Compound E can be treated under known conditions with anisothiocyanate form of a suitable fluorophore (such asfluoresceinisothiocyanate) to form compound F. Compound F can be furthermodified. For example, R¹ can be replaced with a mono, di, ortriphosphate group. Forming a triphosphate can facilitate using thenucleobase containing compounds in enzymatic and template-dependent DNAor RNA synthesis reactions.

Hydrocarbyldithiomethyl-modified nucleotide triphosphates protected atthe 3′ position (as described above) are useful for any sequencingmethod. 3′-hydrocarbyldithiomethyl-modified nucleotide triphosphates canterminate extension of the primer sequence when used in a DNApolymerase-mediated sequencing method. Unlike most conventional dideoxymethods where incorporation of the dideoxynucleotide is permanent,however, termination using a hydrocarbyldithiomethyl-modified nucleotideis reversible. Thus, one of the benefits associated with using ahydrocarbyldithiomethyl-modified nucleotides for sequencing is that thesequencing reaction can be stopped and started by utilizing the labilenature of the protecting group. That is, thehydrocarbyldithiomethyl-moiety can be removed by reducing the disulfidebond of the protecting group. Reducing the disulfide creates an unstableintermediate that spontaneously decomposes to produce a free 3′hydroxyl, which can be used for attaching another nucleotide. Thedisulfide of the hydrocarbyldithiomethyl-moiety can be reduced using anyreducing agent. Suitable reducing agents include dithiothreitol (DTT),mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin,reduced thioredoxin, and any other peptide or organic based reducingagent, or other reagents known to those of ordinary skill in the art.Reduction can be achieved under neutral conditions. It is to beunderstood that the reduction step leading to the spontaneousdecomposition of the intermediate is applicable to allhydrocarbyldithiomethyl-modified compounds. Accordingly, it may benecessary to adjust the conditions of conventional sequencing reactionsusing DNA polymerase enzymes that utilize reduced thioredoxin so thatfree thiols are not present when the hydrocarbyldithiomethyl-modifiednucleotides are added.

The compounds of Formulas 1-3 are useful as reagents in almost anymethod for sequencing a nucleic acid molecule. General methods forsequencing nucleic acids are known and include dideoxy sequencingmethods (Sanger et al., Proc. Natl. Acad. Sci., 74:5463-5467 (1977),chemical degradation methods (Maxam & Gilbert, Proc. Natl. Acad. Sci.,74:560-64 (1977), minisequencing methods (Syvänen et al., Genomics,8:684-92 (1990), and sequencing by synthesis (i.e., multiple iterationsof the minisequencing method). It is common practice in these methods toblock the 3′ hydroxyl of some of the nucleotides. Further, thesequencing by synthesis method requires the availability of nucleotideshaving a reversibly blocked 3′ hydroxyl.

For example, a sequencing method can proceed by contacting a targetnucleic acid with a primer. The target nucleic acid can be any nucleicacid molecule. The primer would also be a nucleic acid molecule.Typically, the primer is shorter than the nucleic acid to be sequenced.Methods for preparing nucleic acids for sequencing and for manufacturingand preparing primer sequences to be used in a sequencing reaction areknown. It is advantageous to design the primer so that at least aportion of the primer is complementary to a portion of the targetnucleic acid. It is also advantageous to design the primer so that thewhole primer is complementary to a portion of the target nucleic acid.

During a sequencing reaction, the primer and target nucleic acidsequences are combined so that the primer anneals or hybridizes to thetarget nucleic acid in a sequence specific manner. A DNA polymeraseenzyme is then used to incorporate additional nucleotides into theprimer in a sequence specific or template-dependent manner such that thenucleotide incorporated into the primer is complementary to the targetnucleic acid. For example, a 3′-hydrocarbyldithiomethyl-modifiednucleotide or a mixture of nucleotides is added to the sequencingreaction at a sufficient concentration so that the DNA polymeraseincorporates into the primer a single hydrocarbyldithiomethyl-modifiednucleotide that is complementary to the target sequence. Theincorporation of the hydrocarbyldithiomethyl-modified nucleotide can bedetected by any known method that is appropriate for the type of labelused.

A second or subsequent round of incorporation for thehydrocarbyldithiomethyl-modified nucleotide can occur after incubatingthe primer target sequence complex with a suitable reducing agent.Further, each round of incorporation can be completed without disruptingthe hybridization between primer and target sequence. After reduction ofthe disulfide, the 3′-OH becomes unblocked and ready to accept anotherround of nucleotide incorporation. The incorporation and reduction stepscan be repeated as needed to complete the sequencing of the targetsequence. In this way, it may be advantageous to differentially labelthe individual nucleotides so that incorporation of differentnucleotides can be detected. Such a method can be used in singlesequencing reactions, automated sequencing reactions, and array basedsequencing reactions.

Hydrocarbyldithiomethyl-modified deoxyribonucleotides andribonucleotides can also be used for synthesizing oligonucleotides.Chemical synthesis of oligoribonucleotides has an added complexitycompared to oligodeoxyribonucleotides due to the presence of the 2′-OHin ribonucleosides. The 2′-OH must be protected during synthesis.Further, the blocking group must be removable during final deblocking.Conditions used for deblocking conventional protecting groups canpromote cleavage and/or migration of intemucleotide linkages, i.e., the5′-3′ linkage of the oligonucleotide may migrate to form a 5′-2′linkage. This cleavage is both acid and base catalyzed, while migrationis acid catalyzed. As such, blocking the 2′-OH with ahydrocarbyldithiomethyl moiety is advantageous because the bond is: 1)stable under conventional/standard acidic and basic conditions whileother blocked regions of the oligoribonucleotide are deprotected, and 2)the hydrocarbyldithiomethyl moiety can be removed under neutralconditions using a simple reducing agent.

A method for synthesizing an oligoribonucleotide using ribonucleosidesmodified at the 2′-OH position with a hydrocarbyldithiomethyl moiety canproceed as follows. A first nucleoside is linked to a solid supportusing known methods. See, e.g., Pon, R T, “Chapter 19 Solid-phaseSupports for Oligonucleotide Synthesis,” Methods in Molecular BiologyVol. 20 Protocols for Oligonucleotides and Analogs, 465-497, Ed. S.Agrawal, Humana Press Inc., Towata, N.J. (1993). The 2′-OH modifyingmoiety can be a hydrocarbyldithiomethyl moiety or any other knownprotecting group (e.g., ester). Alternatively, the first ribonucleosidemonomer is linked to a solid support using known methods but also havinga linker as shown in Formula 6 (described below). It is to be understoodthat the 5′-OH and the 3′-OH are also protected as needed using knownmethods or the methods described herein. After the initial nucleoside istethered to the solid support, additionalhydrocarbyldithiomethyl-modified ribonucleoside monomers are added tothe growing oligoribonucleotide using any of the existing strategies forintemucleotide bond formation. The completed oligoribonucleotide is thendeblocked at all positions except the 2′-O— position by using ammonia.The partially deblocked oligoribonucleotide is then contacted with areducing agent under neutral conditions to achieve the finaldeprotection. Neutral conditions are conditions that do not promotemigration of internucleotide linkages. Conditions having a pH valueranging from about 5 to about 9 and any particular value therebetween,e.g., 7, are considered neutral.

A suitable ribonucleoside for use in a chemical oligonucleotidesynthesis reaction utilizing a hydrocarbyldithiomethyl-modifiedribonucleoside monomer is shown at Formula 4

wherein R¹ is a H or a protecting group, R² is a nucleobase, R⁴, R⁵ andR⁶ are together or separately H or hydrocarbyl. R⁷ may be H,H-phosphonate or phosphoramidite.

An example of Formula 4 is shown at Formula 5

wherein R¹ ² is a nucleobase, R⁴, R⁵ and R⁶ are together or separately Hor hydrocarbyl. Alternatively, other known blocking groups may be usedto block the nucleoside at the 5′-OH and 3′-OH positions according theneeds of the skilled artisan.

Suitable ribonucleosides (as described above) can be prepared usingknown methods and/or the methods described herein for adding ahydrocarbyldithiomethyl moiety to a nucleoside.

Chemical synthesis methods using the oligodeoxyribonucleotides andoligoribonucleotides described herein can include methods for invertingthe orientation of the oligonucleotides on the solid support.International Application WO 98/51698 entitled “Synthesis ofOligonucleotides” discloses methods for preparing immobilizedoligonucleotides and for their subsequent inversion to produceoligonucleotides having a free 3′-OH. These methods together with thecompounds and methods described herein can be used together to produceoligonucleotide arrays. The arrays are useful for binding and sequencingreactions, especially automated sequencing reactions.

Chemical synthesis methods using the oligodeoxyribonucleotides andoligoribonucleotides described herein can include methods for preparingoligonucleotides having different hydrophobic characteristics.Oligonucleotides can be designed to be more or less hydrophobic by usingselectively cleavable protecting groups. To alter the hydrophobiccharacter of the nucleotide, a subset of protecting groups is removedafter synthesizing the oligonucleotide. By altering the ratio ofprotecting groups attached the finished oligonucleotide to the number ofprotecting groups removed, the hydrophobicity of the finishedoligonucleotide can be controlled. These types of oligonucleotides canbe useful as pro-oligonucleotides for antisense drug treatment methodsfor a variety of disease states. The article Tosquellas et al., “ThePro-Oligonucleotide Approach: Solid Phase Synthesis And PreliminaryEvaluation Of Model Pro-Dodecathymidylates,” Nucleic Acids Res. 26:9,2069-74 (1998) provides an example of such pro-oligonucleotides.

Oligonucleotides having altered hydrophobicities can be synthesized byfollowing the methods described herein. For example, a first protectednucleoside is covalently attached to a solid support. Additionalprotected nucleosides are added according to methods described herein toassemble an oligonucleotide. The additional nucleosides may each havedifferent protecting groups. A subset of the protecting groups can beremoved. After reducing the dithiobond, the oligonucleotide is removedfrom the solid phase, washed out and collected. This method allows forisolation of an almost completely protected oligonucleotide. The3′-terminus of the oligonucleotide can be modified with a reactive ordetectable moiety. The oligonucleotide fragments activated at the3′-position can be used for constructing larger oligonucleotides orsynthetic genes. They may also be used in a method for combinatorialsynthesis of gene variants lacking any unwanted stop codons.

Hydrocarbyldithiomethyl-modified compounds may also be used to linkmolecules to a solid support. In particular, it may be advantageous touse hydrocarbyldithiomethyl-modified compounds for chemical synthesis oforganic molecules such as oligonucleotides, peptides, and carbohydrates.Several methods for coupling organic molecules to solid supports areknown. See, e.g., Pon, R T, “Chapter 19 Solid-phase Supports forOligonucleotide Synthesis,” Methods in Molecular Biology Vol. 20Protocols for Oligonucleotides and Analogs, 465-497, Ed. S. Agrawal,Humana Press Inc., Towata, N.J. (1993). Only a few methods, however,provide linkages that are inert under acidic and basic conditions, andyield a free hydroxyl group after cleaving the linkage. For example,photochemically labile o-nitrobenzyl ether linkages, siloxyl linkages,and disiloxyl type linkages that are cleavable using fluoride anions,are both inert under acidic and basic conditions and yield a freehydroxyl group after cleaving the linkage. Use of the above linkages issometimes impractical or associated with unwanted side reactions. Thehydrocarbyldithiomethyl-modified linkages described herein are cleavableunder neutral conditions.

Accordingly, an oligonucleotide synthesis support can include themolecule shown in Formula 6

wherein R¹ is H, phosphate, diphosphate, triphosphate, or a5′-protecting group, R² is a nucleobase, R³ is H, OH, or a protectedform of OH, and Z is a group effective for covalent attachment to asolid support. Examples of Z include amido, ether and any other linkingfunction groups known to those of ordinary skill in the art. Such alinker capable of being coupled to a solid support can be effective forsecuring an oligonucleotide during oligonucleotide synthesis.

An example of a linker described in Formula 6 is shown in Formula 7

wherein R² is a nucleobase, R³ is H, OH, or a protected form of OH.

Chemical synthesis of an oligonucleotide can be done by attaching afirst nucleoside monomer to a solid support. Any known solid support canbe used including non-porous and porous solid supports and organic andinorganic solid supports. Useful solid supports include polystyrenes,cross-linked polystyrenes, polypropylene, polyethylene, teflon,polysaccharides, cross-linked polysaccharides, silica, and variousglasses. In some instances, certain solid supports are not fullycompatible with aspects of oligonucleotide synthesis chemistry. Forexample, strong alkaline conditions at elevated temperatures used fordeprotection of synthetic oligonucleotides or fluoride anions such asthose provided by tetrabutylammonium fluoride cannot be applied tosilica or glass supports. Conventional linkers and methods for attachingmonomers or oligonucleotides to a solid support are known. See Beaucage& Iyer, Tetrahedron, 48(12):2223-2311 (1992).

The invention will be further described in the following examples, whichdo not limit the invention as set forth in the claims.

EXAMPLE 1 Synthesizing 5′-O-FMOC-Thymidine

Thymidine (10 mmol) was dried by coevaporation with dry pyridine (2×30ml), re-dissolved in dry pyridine (50 ml) and cooled using anacetone/carbon dioxide bath to a temperature of −20° C. The thymidinesolution was magnetically stirred and a dichloromethane solution ofFMOC-Cl (12 mmol, 1.2 eq. in 20 ml DCM) was added over a period of 60minutes. The reaction mixture was warmed to room temperature and stirredfor additional 60 minutes. The reaction mixture was partitioned betweensaturated sodium hydrogen carbonate (250 ml) and dichloromethane (3×100ml). The organic phase was saved, combined, evaporated and dried bycoevaporation with toluene (2×50 ml) forming an oily residue. A pureproduct was crystallized from the oily residue using dichloromethane (30ml) and benzene (50 ml) as solvent. Yield 76%—white crystals.

EXAMPLE 2 Synthesizing 5′-O-FMOC-3′-O-methylthiomethyl-thymidine.

The produce of Example 1 (5′-O-FMOC-Thymidine (7.0 mmol)) was dissolvedin 50 ml of an acetic acid:acetic anhydride:DMSO solution (11:35:54,v/v) at 20° C. according to (Zavgorodny et al. (1991) Tetrahedron Lett.32:7593-7596). The solution was stirred at 20° C. for 4 days resultingin a complete conversion of the starting material to methylthiomethylether derivative as monitored by thin layer chromatography (TLC). Thesolvent was evaporated using a rotary evaporator at 50° C. under highvacuum (oil pump). The residue was dissolved in ethanol (30 ml) andpoured into vigorously stirred water (500 ml). A solid materialprecipitated and was filtered off. The precipitate was then dissolved indichloromethane, coevaporated with toluene (2×50 ml), and flashchromatographed using dichloromethane:chloroform (1:1 v:v) as thesolvent to give the final product as an oil. Yield 72%.

EXAMPLE 3 Synthesizing5′-O-FMOC-3′-O-(4-methylphenyltbiosulfonatemethyl)-thymidine.

The product of Example 2 (5′O-FMOC-3′-O-methylthiomethyl-thymidine (4.0mmol)) was dissolved in a solution of dichloromethane (20 ml) andbromine (Br₂) (226 μl) was added at 20° C. After a 10 minute incubation,a potassium salt of p-toluenethiosulfonic acid (10.0 mmol) dissolved indry DMF (10 ml) and lutidine (1.5 ml) was added. The reaction mixturewas stirred for an additional 120 minutes, quenched by addition ofsaturated NaHCO₃ and extracted with dichloromethane (3×50 ml). Theresulting organic phase was evaporated, coevaporated with toluene, andflash chromatographed using chloroform as the final solvent. The finalproduct was isolated as an oil. Yield 58%.

EXAMPLE 4 Synthesizing 3′-O-hydrocarbyldithiomethyl)thymidineDerivatives.

The product of Example 3 (1 mmol) is dissolved in pyridine (5.0 ml) andan appropriate thiol, such as reduced cystamine (“R”SH, i.e.,hydrocarbylthiol) (1.1 mmol) dissolved in pyridine (2.0 ml), is added.

The mixture is stirred for 60 min at 20° C., then extracted usingconventional bicarbonate extraction methods and purified by flashchromatography. In some instances it may be advantageous to continue thesynthetic process by addition of dry triethylamine (4.0 mmol) in orderto remove the 5′-O-FMOC protecting group. After 45 minutes the solventis evaporated and the 5′-OH derivative is isolated by chromatographyafter the standard work-up using aqueous NaHCO₂ and dichloromethane andevaporating the organic extracts.

EXAMPLE 5 Synthesizing 3′-O-(2-N-dansylethyldithiomethyl)-thymidine.

The general procedure of Example 4 is followed using N-dansylethanethiolas the thiol. N-dansylethanethiol is prepared by reacting cystaminedihydrochloride with dansyl chloride followed by reducing the disulfidewith sodium borohydride. N-dansylethanethiol is isolated using rapidsilica gel purification.

After forming the desired 2-N-dansylethyldithiomethyl linkage, the 5′FMOC group is removed using known methods.

EXAMPLE 6 Synthesizing3′-O-(2-N-dansylethyldithiomethyl)-thymidine-5′-triphosphatetetralithium Salt.

The product of Example 5 (3′-O-(2-N-dansylethyldithiomethyl)-thymidine(0.1 mmol)) is dried by coevaporation with dry pyridine (2×5 ml) anddissolved in dry acetonitrile (2.0 ml). Phosphorotristriazolide (0.1 M)in dry acetonitrile is prepared from phosphorus oxychloride and triazoleas described in Kraszewski & Stawinski, Tetrahedron Lett., 21:2935-2936(1980). Phosphorotristriazolide (1.5 ml, 1.5 eq.) is added to the3′-O-(2-N-dansylethyldithiomethyl)-thymidine at room temperature or 20°C. The mixture is stirred for 5 minutes at which point n-butylammoniumpyrophosphate in dry DMF (0.2 M, 1.5 ml, 2.0 eq.) is added. The mixtureis stirred overnight at 20° C. Water (2 ml) is then added and hydrolysisof the phosphates occurs (180 min). The nucleotide triphosphate isapplied to an anion exchange column Mono Q (TM) (Pharmacia Biotech.Sweden) equilibrated with triethylammonium bicarbonate (TEAB) (0.01 M)and eluted from the Mono Q (TM) column using a linear gradient of TEAB(0.85M):acetonitrile (33%, v/v). The isolated3′-O-(2-N-dansylethyldithiomethyl)-thymidine-5′-triphosphatetetralithium salt fraction is evaporated, coevaporated with water andpassed through a Dowex 50W×8 (BDH) in a lithium form to accomplishexchange of the triethylamnionium to the lithium ions. At this point thehydrocarbyldithiomethyl-modified nucleotide is ready to be used.

Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1. A hydrocarbyldithiomethyl-modified compound comprising the formula:

or a salt thereof, wherein R¹ is H, a protecting group, phosphate,diphosphate, triphosphate, or residue of a nucleic acid; R² is anucleobase; R³ is H, OH, or a protected form of OH; and R⁴, R⁵ and R⁶are together or separately H, hydrocarbyl, or a residue of a solidsupport.
 2. The compound of claim 1 wherein R⁴, R⁵ and R⁶ together orseparately further comprise a labeling group.
 3. The compound of claim 1wherein R⁴, R⁵ and R⁶ comprise together or separately an electrondonating or withdrawing function.
 4. The compound of claim 3 whereinsaid electron donating or withdrawing function contains a heteroatomselected from the group consisting of oxygen, nitrogen, sulfur, andsilicon.
 5. The compound of claim 1 wherein R⁴, R⁵ and R⁶ are togetheror separately H, methyl, ethyl, isopropyl, t-butyl, phenyl, or benzyland wherein either R⁴, R⁵ and R⁶ is modified with a labeling group.
 6. Acompound comprising the Formula:

or a salt thereof, wherein R¹ is H, a protecting group, a phosphate,diphosphate, or a triphosphate, or a residue of a nucleic acid; R² is anucleobase; R³ is H or OH, or a protected form of OH; and R⁴ is H orhydrocarbyl.
 7. The compound of claim 6 wherein R⁴ is modified with alabeling group.
 8. The compound of claim 6 wherein R⁴ comprisesnitrogen.
 9. The compound of claim 6 wherein R⁴ is covalently linked toa solid support.